北京大学使用我司磁珠泛素和泛素化蛋白纳米受体发表文章

Construction of nano receptors for ubiquitin and ubiquitinated proteins based on the region-specific interactions between ubiquitin and polydopamine†

基于泛素和聚多巴胺区域特异性相互作用构建泛素和泛素化蛋白纳米受体
Zezhou Li,‡a   Xinyi Li,‡a   Wei Xian,‡b   Huaisyuan Xie,a   Ying Sun,a   Yuxuan Zhang,a   Jiayu Wang,a   Hongwei Li,a   Changwen Jin,a   Xiaoyun Liu,*b   Zhiwei Zhu 

Abstract
Ubiquitination is a prevalent post-translational modification that controls a multitude of important biological processes. Due to the low abundance of ubiquitinated proteins, highly efficient separation and enrichment approaches are required for ubiquitinome analysis. In this work, we disclose the region-specific interactions between the hydrophobic patch of ubiquitin and polydopamine. Taking advantage of this inherent binding property, we have constructed surface-imprinted magnetic nanoparticles (NPs) for ubiquitin by sequential dopamine polymerization and surface PEGylation. The obtained molecularly imprinted polymer (MIP) NPs showed a binding constant of 2.6 × 106 L mol−1 for the template ubiquitin. The bound ubiquitin could be quantitatively released by heating to 70 °C at pH 2.0 or 90 °C at neutral (pH 7.0) conditions. The MIP NPs exhibited nano receptor-like property which not only effectively blocked the formation of branched ubiquitin chains but also selectively separated ubiquitin from the bacterial cell lysates. By incubating the MIP NPs with the lysates of 293T cells, totally 529 ubiquitinated proteins were captured, among which 287 proteins were not identified by the anti-ubiquitin monoclonal antibodies (mAbs). With the distinct merits of low cost and high stability, the as-prepared MIP NPs may be utilized either separately or as an important complement to the mAbs for the purification and enrichment of ubiquitin and ubiquitinated proteins from complex biological samples. Furthermore, due to the flexibility in modification of the binding sites during or after the imprinting reactions, the results of this work also paved the way for generation of artificial receptors for branched ubiquitin chains and polyubiquitinated proteins with higher avidity and specificity.

泛素化是一种普遍的翻译后修饰，它控制着许多重要的生物过程。由于泛素化蛋白质的丰度低，泛素组分析需要高效的分离和富集方法。在这项工作中，我们揭示了泛素疏水贴片和聚多巴胺之间的区域特异性相互作用。利用这种固有的结合特性，我们通过顺序多巴胺聚合和表面聚乙二醇化构建了泛素的表面印迹磁性纳米粒子 (NPs)。获得的分子印迹聚合物 (MIP) NPs 对模板泛素的结合常数为 2.6 × 106 L mol-1。结合的泛素可以通过在 pH 2.0 下加热至 70 °C 或在中性 (pH 7.0) 条件下加热至 90 °C 来定量释放。 MIP NPs表现出类似纳米受体的特性，不仅有效地阻止了分支泛素链的形成，而且还选择性地将泛素从细菌细胞裂解物中分离出来。通过将 MIP NPs 与 293T 细胞的裂解物一起孵育，共捕获了 529 个泛素化蛋白质，其中 287 个蛋白质未被抗泛素单克隆抗体 (mAb) 鉴定。由于具有低成本和高稳定性的独特优点，所制备的 MIP NP 可以单独使用，也可以作为 mAb 的重要补充，用于从复杂的生物样品中纯化和富集泛素和泛素化蛋白质。此外，由于在印迹反应期间或之后修饰结合位点的灵活性，这项工作的结果也为产生具有更高亲和力和特异性的分支泛素链和多泛素化蛋白的人工受体铺平了道路。